表达胰高血糖素样肽1重组腺伴随病毒载体的构建  

Construction of a recombinant adeno-associated virus plasmid expressing glucagon-like peptide-1

作　　者：娄明武[1] 梁冰[1] 张明东[1] 杨广笑 王全颖 

机构地区：[1]深圳市龙岗中心医院介入影像科,广东省深圳市518116 [2]西安市华广生物工程有限公司,陕西省西安市710025

出　　处：《中国组织工程研究与临床康复》2011年第28期5299-5302,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省科学技术厅资助项目(2005B36001082);深圳市科技和信息局资助项目(JH200505260306A);深圳市龙岗区委组织部2008年专家提升计划资助项目~~

摘　　要：背景:胰高血糖素样肽1(glucagon-like peptide-1,GLP-1)在人体内半衰期过短限制了其应用。目的:构建可表达GLP-1的重组腺伴随病毒。方法:将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中,构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒。采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系,用其感染Hela细胞。结果与结论:重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342bp的目的片段,说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内。免疫细胞化学结果显示,转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒,阳性率达到70%以上,说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1。BACKGROUND：Glucagon-like peptide-1 （GLP-1） is limited by half-life in human body. OBJECTIVE：To construct a recombinant adeno-associated virus vector expressing GLP-1. METHODS：The fusion gene NT4-GLP-1 was inserted into the adeno-associated virus packaging plasmid pSSHG-CMV to generate the adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. 80% of fused 293 cells were co-transfected by using the calcium phosphate precipitation method, with aid packaging plasmids pAAV/Ad, pFG140 and the recombinant adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. Virus effects were confirmed by immunohistochemistry at the same time. RESULTS AND CONCLUSION：Restriction enzyme EcoRⅠidentification showed that the recombinant plasmid pSSHG/NT4-GLP-1 contained a 342 bp target segment, indicating that the fusion gene NT4-GLP-1 has been successfully transfected into recombinant adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. Immunocytochemical staining results showed that Hela cells transfected by plasmid pSSHG/NT4-GLP-1 contained a large number of brown yellow particles, with the rate of positive cells more than 70%. These findings suggest that adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1 expressed GLP-1.

关 键 词：胰高血糖素样肽1 腺伴随病毒 重组质粒 构建 组织构建 

分 类 号：R318[医药卫生—生物医学工程]